Glycosyltransferase glycosylating flavokermesic acid and/or or kermesic acid

ABSTRACT

An isolated glycosyltransferase (GT) polypeptide capable of: (I): conjugating glucose to flavokermesic acid (FK); and/or (II): conjugating glucose to kermesic acid (KA) and use of this GT to e.g. make Carminic acid.

FIELD OF THE INVENTION

The present invention relates to an isolated glycosyltransferase (GT)polypeptide capable of: (I): conjugating glucose to flavokermesic acid(FK); and/or (II): conjugating glucose to kermesic acid (KA) and the useof this GT to make e.g. Carminic acid.

BACKGROUND OF THE INVENTION

The natural pigment carminic acid is one of the most frequently usedcolorants of food, medicine, cosmetics and textiles.

Carminic acid is a colorant, which can be extracted from the femaleinsect bodies of Dactylopius coccus costa (alternative name Coccus cactiL.). The insects live on Nopalea coccinellifera, Opuntia fidus indicaand other plants of the family Cactaceae cultivated for instance in thedesert areas of Mexico, Central and South America and Canary Islands.Depending on the pH the colorant may be a color in a spectrum fromorange over red to purple and is generally known as cochineal orcochineal color. Carmine colorant is widely used in foods and beverages.

As known in the art Porphyrophora polonica is also producing carminicacid and was cultured for production of carminic acid in e.g. Poland.

In relation to current industrial relevant production, carminic acid isharvested by extraction from the insect's dried bodies with water oralcohol.

The insects (Dactylopius coccus) are cultured on cacti and the supplymay therefore be relatively expensive and subject to undesirablevariations and price fluctuations.

In order to try to resolve the problem of undesirable variations andprice fluctuations—U.S. Pat. No. 5,424,421 (European Colour, published1995) describes chemical synthesis of carminic acid by a route ofsynthesis involving different intermediates.

As discussed in e.g. WO02006/056585A1 (Chr. Hansen A/S)—during theaqueous based extraction of carminic acid from the insect, an amount ofinsect protein is also released from the insect and will be contained inthe color extract and it has been reported that the cochineal insectproteins could create some allergy related problems. In WO02006/056585A1a special process to reduce the amount of insect protein from the insectextract solution is described—however, the final produced colorcomposition/product of WO02006/056585A1 will still comprise some amountsDactylopius coccus costa insect proteins.

The structure of carminic acid is shown in FIG. 1—as can be seen it is aso-called C-glucoside (i.e. wherein the glucose is joined/conjugated tothe aglucon by a carbon-carbon linkage).

According to the art—the term “aglycon” denotes the non-carbohydratepart of the corresponding glycosylated form of the aglycon. When thesugar is glucose the aglycon may be termed aglucon.

According to the art—the term “glycoside” denotes a compound which byhydrolysis results in a sugar and a non-sugar (aglycon) residue, e.g.glucosides can give glucose, galactosides can give galactose. As shownin FIG. 1—hydrolysis of the C-glucoside carminic acid results in glucoseand the aglucon kermesic acid (KA).

The in vivo insect (Dactylopius coccus) biosynthetic pathway involved incarmine production is currently not described in details—accordingly,based on the prior art the skilled person does not know which compoundis the aglucon during the in vivo Dactylopius coccus biosyntheticproduction of carminic acid.

Analysis of D. coccus has shown that a broad range of compounds relatedto carminic acid are present in extract from D. coccus and numerous ofthese compounds could in principle be glucosylated during the in vivoDactylopius coccus biosynthetic production of carminic acid.

For instance, the article of Stathopoulou et al (Analytica Chimica Acta804 (2013) 264-272) describes six new anthraquinones that are present inextract from D. coccus and any of these six new anthraquinones (see e.g.FIG. 1 of the article) could in principle be the molecule which isglucosylated during the in vivo Dactylopius coccus biosyntheticproduction of carminic acid.

Furthermore, and as known in the art, the primary glucosylated compoundformed during the in vivo biosynthetic production of the glucoside endproduct may be an unstable intermediate compound that will not beidentified in an isolated extract from D. coccus as e.g. analyzed in theabove discussed article of Stathopoulou et al.

Based on the prior art, it could be speculated that a relevant primaryglucosylated compound during the in vivo Dactylopius coccus biosyntheticproduction of carminic acid could e.g. be an unstable intermediatepolyketide compound with around the same number of carbon atoms as e.g.flavokermesic acid.

According to the art—the term “glycosyltransferase” (GT) denotes aglycosyltransferase enzyme capable of transferring a sugar from anactivated nucleotide sugar to an aglycon to form a glycoside.

A herein relevant DNA or amino acid sequence of a glycosyltransferaseinvolved in the in vivo insect (Dactylopius coccus) biosynthetic pathwayof carminic acid is not explicitly described in the prior art.

As known in the art, for insects that accumulate low molecular weightchemicals the relevant biosynthetic pathway genes are sometimes notpresent in the insect genome. For instance, some insects take upglycosides from the plants they feed on—see e.g. the article ofZagrobelny et al (Cyanogenic glucosides and plant-insect interactions;Phytochemistry. 2004 February; 65(3):293-306) or the article of Geuderet al (Journal of Chemical Ecology, Vol. 23, No. 5, 1997). Also, therelevant biosynthetic pathway genes are sometimes found in themicroorganisms living in the insects, see e.g. the article of Genta etal, (Potential role for gut microbiota in cell wall digestion andglucoside detoxification in Tenebrio molitor larvae), Journal of InsectPhysiology 52 (2006) 593-601.

Dactylopius coccus insects feed on cactus plants and it could be that D.coccus insects (like other insects) take up relevant glycosides from thecactus they feed on

Accordingly, based on the prior art the skilled person could not know ifthe genome of Dactylopius coccus actually would comprise a gene encodinga glycosyltransferase involved in the in vivo biosynthetic pathwayleading to carminic acid.

WO2004/111254A1 (Poalis A/S) describes in vivo production of aglucosylated form of vanillin in e.g. eukaryotic cell yeast cells and/orprokaryotic E. coli cells by using a glucosyltransferase for conjugatingglucose to the vanillin aglucon in vivo within a microorganism cell.Natural vanillin is obtained from the plant vanilla bean. Accordingly,in the prior art successful in vivo production has been described inmicroorganism cells of plant glycoside compounds (such as e.g. vanillinglucoside).

SUMMARY OF THE INVENTION

The problem to be solved by the present invention relates to theprovision of a glycosyltransferase (GT) involved in a biosyntheticpathway that may lead to carminic acid and the use of thisglycosyltransferase to make e.g. carminic acid.

As discussed in working examples herein—the present inventors sequencedthe entire genome and transcriptome (i.e. set of RNA molecules includingmRNA) of Dactylopius coccus and microbial symbionts.

The identified oligonucleotide sequences obtained from the genome andtranscriptome were analyzed for similarity to public knownC-glycosyltransferase sequences and the result was negative. None of theidentified gene sequences of the genome/transcriptome showed significantsimilarity to publicly known C-glycosyltransferase sequences.

As discussed above—based on the prior art the skilled person could notknow if the genome of Dactylopius coccus actually would comprise a geneencoding a glycosyltransferase involved in the in vivo biosyntheticpathway leading to carminic acid. However, the present inventorscontinued to investigate the matter.

As discussed in working examples herein—the present inventors identifieda Dactylopius coccus extract (including extracts of the endosymbiontspresent in D. coccus) with relevant GT activity and by a combination ofrelevant purification and testing steps the inventors were finally ableto obtain a relatively pure fraction/composition wherefrom it waspossible to obtain several partial amino acid sequences of putative GTenzyme candidates.

The partial amino acid sequences of these enzyme candidates werecompared to the identified gene sequences of the transcriptome and afterfurther detailed work, a sequence encoding a glycosyltransferase enzymesequence was identified—the polynucleotide sequence encoding thisisolated/cloned novel glycosyltransferase is shown in SEQ ID NO: 1 andthe polypeptide amino acid sequence is shown in SEQ ID NO: 2.

The glycosyltransferase enzyme of SEQ ID NO: 2 may be termed “DcUGT2”.

It is believed that the described isolated/cloned glycosyltransferase isthe first described insect derived glycosyltransferase. As described,the identified gene sequences of the Dactylopius coccus transcriptomewere analyzed for similarity to relevant public knownglycosyltransferase sequences and the result was negative.

The present inventors found, that the publicly known prior artglycosyltransferase sequences have less than 45% identity to the novelglycosyltransferase polypeptide sequence shown as SEQ ID NO: 2.

As discussed in working examples herein—the present inventors tested theactivity of the isolated/cloned novel glycosyltransferase and found thatit was able to conjugate glucose to the aglycons flavokermesic acid (FK)and kermesic acid (KA)—see FIG. 1. Analysis of the glucosylated productshowed that among the many potential O- and C-glucoside productspotentially formed only a single glucoside product was formed with eachof the two substrates. The analysis showed that each of the productswere C-glucosylated at position 7 of the anthraquinone structure, moreprecisely7-α-D-Glucopyranosyl-9,10-dihydro-3,6,8-trihydroxy-1-methyl-9,10-dioxoanthracenecarboxylicacid (DcII—see FIG. 1) and7-α-D-Glucopyranosyl-9,10-dihydro-3,5,6,8-tetrahydroxy-1-methyl-9,10-dioxoanthracenecarboxylicacid (carminic acid—see FIG. 1) by the GT when UDP-glucose was used asthe sugar donor substrate.

The article of Gutmann et al (Pure Appl. Chem, 2013 Jul. 9) describesthat even though a number of C-glycosides have been isolated fromnatural sources, the enzymes responsible for their biosynthesis are onlyknown in very few cases, and the biocatalytic approaches for C-glycosideproduction has yet to be established

The article of Baig et al (Angew Chem Int Ed Engl. 2006 Nov. 27;45(46):7842-6) describes the glycosyltransferase (GT) termed UrdGT2 andexplains that it is able to conjugate a sugar to a number of aglyconsthat may herein be considered relatively similar to flavokermesic acid(FK) and kermesic acid (KA).

Accordingly, it may be said that UrdGT2 prima facie would be a qualifiedguess for a GT that could be capable of conjugating sugar toflavokermesic acid (FK) and/or kermesic acid (KA).

As discussed in working Example herein—the present inventors cloned theUrdGT2 and tested it for flavokermesic acid (FK) and/or kermesic acid(KA) GT activity and it was found that the UrdGT2 was able to useUDP-glucose as a sugar donor, but UrdGT2 did not glucosylate any of thetested putative aglycons—i.e. no GT activity was identified in relationto these aglycons.

The UrdGT2 has around 15-20% amino acid identity with SEQ ID NO:2disclosed herein.

Based on both publicly known GT sequences and not publicly known GTsequences—the present inventors made different detailed sequencealignment investigations.

Based on these sequence alignment investigations—it is believed thatamino acids 20 to around 468 of SEQ ID NO:2 comprise the essential partsof the catalytic domain. Based on these sequence alignmentinvestigations—it is believed that amino acids from around 291 to around383 of SEQ ID NO:2 comprise the so-called activated nucleotide sugarbinding site.

Based on these sequence alignment investigations—it is believed thatamino acids from around 1 to around 20 of SEQ ID NO:2 comprise theso-called signal peptide and it is believed that this signal peptide maybe removed without significantly affecting the herein relevant GTactivity of the enzyme.

Furthermore, it is believed that the activated nucleotide sugar bindingsite may be substituted by similar (e.g. prior art known) GT activatednucleotide sugar binding site sequences—such as e.g. the activatednucleotide sugar binding site as described in Radominska-Pandya A,Bratton S M, Redinbo M R, Miley M J. Drug Metab Rev. 2010 February;42(1):133-44) and Plant Physiology, November 2008, Vol. 148, pp.1295-1308.

Accordingly, a first aspect of the present invention relates to anisolated glycosyltransferase polypeptide capable of:

-   -   (I): conjugating glucose to flavokermesic acid (FK); and/or    -   (II): conjugating glucose to kermesic acid (KA);        and wherein the glycosyltransferase polypeptide is at least one        polypeptide selected from the group consisting of:    -   (a) a polypeptide comprising an amino acid sequence which has at        least 70% identity with amino acids 1 to 515 of SEQ ID NO:2;    -   (b) a polypeptide comprising an amino acid sequence which has at        least 70% identity with amino acids 20 to 468 of SEQ ID NO:2;    -   (c) a polypeptide which is encoded by a polynucleotide that        hybridizes under at least medium stringency conditions with (i)        nucleotides 1 to 1548 of SEQ ID NO:1 or (ii) a complementary        strand of (i); and    -   (d) a fragment of amino acids 1 to 515 of SEQ ID NO:2, which has        the glycosyltransferase activity as specified in (I) and/or        (II).

As understood by the skilled person in the present context—the term “aglycosyltransferase polypeptide capable of” of the first aspect relatesto that the glycosyltransferase shall be capable of performing theglycosyltransferase (I) and/or (II) activity—but it may or may not alsobe capable of performing other glycosyltransferase activities.

As understood by the skilled person in the present context—thedisclosure of the herein described novel glycosyltransferase sequence isan important tool to identify similar glycosyltransferases in e.g. otherinsects than Dactylopius coccus and without being limited to theory—itis believed that a sequence with at least 70% identity with SEQ ID NO:2would be a plausible good candidate for a another herein relevantglycosyltransferase.

A second aspect of the present invention relates to an isolatedpolynucleotide comprising a nucleotide sequence which encodes thepolypeptide of the first aspect and/or herein relevant embodimentsthereof.

A third aspect of the present invention relates to a nucleic acidconstruct comprising the isolated polynucleotide of the second aspectand/or herein relevant embodiments thereof operably linked to one ormore control sequences that direct the production of the polypeptide inan expression host.

A fourth aspect of the present invention relates to a recombinantexpression vector comprising the nucleic acid construct of the thirdaspect and/or herein relevant embodiments thereof.

A fifth aspect of the present invention relates to a recombinant hostcell comprising the nucleic acid construct of the third aspect and/orherein relevant embodiments thereof.

As discussed above—based on the prior art the skilled person does notknow which compound is the primary glycosylated compound during thebiosynthetic production of carminic acid in vivo in Dactylopius coccus.

It has been shown that D. coccus contains a GT able to C-glycosylateflavokermesic acid (FK) and/or kermesic acid (KA).

It is evident that this important knowledge is sufficient in order toe.g. produce carminic acid without the need to make an extraction frominsects and thereby be able to make a carminic acid colorcomposition/product essentially free of e.g. unwanted Dactylopius coccuscosta insect proteins.

Since the skilled person did not know which compound is glycosylatedduring the in vivo Dactylopius coccus biosynthetic production ofcarminic acid it was actually unknown to the skilled person if there innature actually existed a glycosyltransferase capable of C-glycosylatingflavokermesic acid aglycon and/or the kermesic acid aglycon.

It is believed that the herein disclosed novel glycosyltransferaserepresents the first isolated glycosyltransferase capable ofglycosylating flavokermesic acid aglycon and/or kermesic acid.

Accordingly, based on the technical disclosure herein—it is believedthat the skilled person would be able to identify other suitableglycosyltransferases capable of glycosylating flavokermesic acid (FK)and/or kermesic acid (KA).

The skilled person would appreciate that one way to try to identify ifan organism/plant would comprise a relevant glycosyltransferase would beto contact relevant aglycons (i.e. FK and/or KA) to the organism/plant(in vivo and/or in vitro) and then measure if the organism/plantproduces relevant FK and/or KA glycosides.

As understood herein, if the organism/plant produces relevant FK and/orKA glycosides then the organism/plant will comprise a relevantglycosyltransferase—i.e. a glycosyltransferase which is capable ofglycosylating flavokermesic acid in order to produce flavokermesic acidglycoside; and/or capable of glycosylating kermesic acid in order toproduce kermesic acid glycoside.

As discussed below—based on the strategy above, the present inventorsfound that relevant glycosyltransferases may be identified in Aloeplants, Haworthia plants, Sorghum or rice plants.

Accordingly, a sixth aspect of the present invention relates to a methodfor producing flavokermesic acid (FK) glycoside and/or kermesic acid(KA) glycoside, wherein the method comprises following steps:

(A): contacting in vitro or in vivo in a recombinant host cellcomprising a glycosyltransferase gene encoding a glycosyltransferase:

-   -   (a1): flavokermesic acid (FK) with a glycosyltransferase capable        of glycosylating the flavokermesic acid under suitable        conditions wherein there is produced the flavokermesic acid        glycoside; and/or    -   (a2): kermesic acid (KA) with a glycosyltransferase capable of        glycosylating the kermesic acid under suitable conditions        wherein there is produced the kermesic acid glycoside.

The term “recombinant host cell” should herein be understood accordingto the art. As known in the art, recombinant polynucleotide (e.g. DNA)molecules are polynucleotide (e.g. DNA) molecules formed by laboratorymethods of genetic recombination (such as molecular cloning) to bringtogether genetic material from multiple sources, creating sequences thatwould not otherwise be found in biological organisms. As understood bythe skilled person—a recombinant host cell comprises recombinantpolynucleotide (e.g. DNA) molecules and a recombinant host cell willtherefore not be understood as covering a natural wildtype cell assuch—such as e.g. a natural wildtype Dactylopius coccus cell.

Said in other words and as understood by the skilled person—for instancea natural wildtype Dactylopius coccus cell as such does not contain arecombinant glycosyltransferase gene encoding a glycosyltransferase.

It may be preferred that the recombinant host cell in step (A) is arecombinant host cell comprising a recombinant glycosyltransferase geneencoding a glycosyltransferase

As discussed herein—in working Examples there was made a contacting invitro of flavokermesic acid (FK) and/or kermesic acid (KA) with theglycosyltransferase of SEQ ID NO:2. It may be seen as routine work forthe skilled person to perform such an in vitro contacting step.

The glycosyltransferase of SEQ ID NO:2 was recombinantly expressed in ayeast cell (see working Example)—accordingly, a recombinant yeast hostcell comprising a recombinant glycosyltransferase gene encoding aglycosyltransferase of SEQ ID NO:2 was made.

It is believed that if flavokermesic acid (FK) and/or kermesic acid (KA)would be added under suitable conditions to a fermentation medium the FKand/or KA compound(s) would enter into e.g. yeast cells fermented in themedium—accordingly, if e.g. the yeast cells are recombinant yeast hostcells comprising a recombinant glycosyltransferase gene encoding aglycosyltransferase then there would be made a contacting in vivo in arecombinant host cell of FK and/or KA with a glycosyltransferase.

In e.g. above discussed WO2004/111254A1 (Poalis A/S) such in vivocontacting of different aglycon compounds in different recombinant hostcells were made and the skilled person would know how to perform suchcontacting in vivo in a recombinant host cell of a relevant aglycon(here flavokermesic acid (FK) and/or kermesic acid (KA)) and arecombinantly expressed glycosyltransferase.

As discussed above—it is believed that the herein disclosed novelglycosyltransferase represents the first time that there has beendisclosed an isolated glycosyltransferase capable of glycosylatingflavokermesic acid aglycon and/or kermesic acid aglycon.

It is believed that relevant partial sequences of herein disclosed novelglycosyltransferase of SEQ ID NO:2 may be recombinantly introduced intoanother glycosyltransferase sequence in order to construct a new hybridglycosyltransferase sequence capable of glucosylating flavokermesic acidand/or kermesic acid. Such GTs with reduced k_(M) or increased V_(max)may prove important in securing rapid glucosylation of the substratesthat may show toxic effects inhibiting yeast growth if accumulating athigh levels (Esben Halkjaer Hansen et al. Phytochemistry 70(4):473-482). Likewise, if so desired it is envisioned possible to modifythe substrate specificity towards glucosylation of earlier pathwayintermediates.

Accordingly, a further aspect of the present invention relates to amethod for constructing a novel isolated hybrid glycosyltransferasepolypeptide capable of:

-   -   (I): conjugating glucose to flavokermesic acid (FK); and/or    -   (II): conjugating glucose to kermesic acid (KA),        wherein the method comprises following steps:    -   (i): inserting a polynucleotide sequence encoding a fragment of        an amino acid sequence which has at least 70% identity with        amino acids 1 to 515 of SEQ ID NO:2 (preferably a fragment of an        amino acid sequence which has at least 90% identity with amino        acids 1 to 515 of SEQ ID NO:2, more preferably a fragment of an        amino acid sequence which has at least 99% identity with amino        acids 1 to 515 of SEQ ID NO:2) wherein the fragment comprises at        least 75 amino acids (preferably at least 100 amino acids, more        preferably at least 150 amino acids and even more preferably at        least 468 amino acids), into another polynucleotide sequence        derived from a glycosyltransferase in order to thereby construct        a novel recombinant hybrid polynucleotide sequence;    -   (ii): expressing the novel hybrid polypeptide which is encoded        by the novel recombinant hybrid polynucleotide sequence of step        (i);    -   (iii): isolating the expressed novel hybrid polypeptide of step        (ii);    -   (iv): testing if the isolated novel hybrid polypeptide of        step (iii) is capable of:        -   (I): conjugating glucose to flavokermesic acid (FK); and/or        -   (II): conjugating glucose to kermesic acid (KA); and    -   (v) if positive in test of step (iv) then has been constructed        the novel isolated hybrid glycosyltransferase polypeptide        capable of:        -   (I): conjugating glucose to flavokermesic acid (FK); and/or        -   (II): conjugating glucose to kermesic acid (KA).

Definitions

All definitions of relevant terms are in accordance with what would beunderstood by the skilled person in relation to the relevant technicalcontext.

The term “aglycon” denotes non-carbohydrate part of the correspondingglycosylated form of the aglycon. When the sugar is glucose the aglyconmay be termed aglucon. Further, when the sugar is glucose the termglucosylated may be used instead of glycosylated.

When the aglycon is glycosylated at a hydroxy group there is generallycreated a so-called O-glycosidic bond—i.e. a so-called O-Glycoside (orO-Glucoside if the sugar is glucose). When the aglycon is glycosylatedby a carbon-carbon linkage it may be termed a C-glycosidic bond—i.e. aso-called C-Glycoside (or C-Glucoside if the sugar is glucose).

The term “glycoside” denotes a compound which on hydrolysis can give asugar and a non-sugar (aglycon) residue, e.g. glucosides can giveglucose and galactosides can give galactose.

The term “glycosyltransferase” denotes an enzyme capable of conjugatinga nucleotide activated sugar to a compound (e.g. an aglycon compound).The sugar may e.g. be D and L isomers of galactose, glucosamine,N-acetylglusamine, xylose, glucuronic acid, rhamnose, arabinose, mannoseor glucose. Alternatively the sugar may be a carbohydrate derivativesuch as e.g. inositol, olivose, rhodinose and etc available asnucleotide diphosphates. Further the sugar may e.g. be a monosaccharide,a disaccharide or a trisaccharide. In the case of oligo- andpolysaccharides the sugars are linked one by one, by e.g. involving useof one or several glycosyltransferases. If the sugar is glucose theglycosyltransferase may be termed a glucosyltransferase.

When the glycosyltransferase conjugates a nucleotide activated sugar toa compound via a C-glycosidic bond it may be termed aC-glycosyltransferase.

When the glycosyltransferase conjugates a sugar to an aglycon via anO-glycosidic bond it may be termed an O-glycosyltransferase.

The term “hybridizes” in relation to a polynucleotide which hybridizesunder at least medium stringency conditions with (i) nucleotides 1 to1548 of SEQ ID NO:1 or (ii) a complementary strand of (i) relates to thenucleotide sequence which hybridizes to a labeled nucleic acid probecorresponding to the nucleotide sequence shown in SEQ ID NO:1 or itscomplementary strand under medium to very high stringency conditions.Molecules to which the nucleic acid probe hybridizes under theseconditions can be detected using e.g. X-ray film.

Herein relevant hybridization stringency conditions are stringencyconditions that the skilled person normally would understand arerelevant—i.e. for medium stringency conditions the conditions thatskilled person would understand are medium stringency conditions. Theskilled person knows relevant hybridization stringency conditions—seee.g. (J. Sambrook, E. F. Fritsch, and T. Maniatus, 1989, MolecularCloning, A Laboratory Manual, 2d edition, Cold Spring Harbor, N.Y.).

According to the art—for long probes of at least 100 nucleotides inlength, very low to very high stringency conditions are defined asprehybridization and hybridization at 42° C. in 5×SSPE, 0.3% SDS, 200μg/ml sheared and denatured salmon sperm DNA, and either 25% formamidefor very low and low stringencies, 35% formamide for medium andmedium-high stringencies, or 50% formamide for high and very highstringencies, following standard Southern blotting procedures for 12 to24 hours optimally.

For long probes of at least 100 nucleotides in length, the carriermaterial is finally washed three times each for 15 minutes using 2×SSC,0.2% SDS preferably at least at 45° C. (very low stringency), morepreferably at least at 50° C. (low stringency), more preferably at leastat 55° C. (medium stringency), more preferably at least at 60° C.(medium-high stringency), even more preferably at least at 65° C. (highstringency), and most preferably at least at 70° C. (very highstringency).

The term “in vitro” (Latin: in glass) relates to studies that areconducted using components of an organism that have been isolated fromtheir usual biological surroundings in order to permit a more detailedor more convenient analysis than can be done with whole organisms. Theseexperiments are commonly called “test tube experiments”. In contrast, invivo studies are those that are conducted with living organisms in theirnormal intact state.

The term “in vivo” (Latin for “within the living”) relates toexperimentation using a whole, living organism as opposed to a partialor dead organism, or an in vitro (“within the glass”, e.g., in a testtube or petri dish) controlled environment.

The term “isolated polynucleotide” essentially relates herein to thatthe polynucleotide is isolated from its natural environment—said inother words that the polynucleotide preparation is essentially free ofother polynucleotide material with which it is natively associated. Thepolynucleotide sequence encoding the herein described isolated/clonednovel glycosyltransferase is shown in SEQ ID NO: 1 and it was isolatedfrom the insect Dactylopius coccus. Accordingly, as understood by theskilled person—the term isolated polynucleotide does not cover thepolynucleotide of SEQ ID NO: 1 when it is naturally present in thegenome of Dactylopius coccus. The term “isolated polynucleotide”essentially relates to that the isolated polynucleotide is in a formsuitable for use within genetically engineered protein productionsystems. Thus, an isolated polynucleotide contains at most 10%,preferably at most 8%, more preferably at most 6%, more preferably atmost 5%, more preferably at most 4%, more preferably at most 3%, evenmore preferably at most 2%, most preferably at most 1%, and even mostpreferably at most 0.5% by weight of other polynucleotide material withwhich it is natively associated. The term “isolated polynucleotide” mayherein alternatively be termed “cloned polynucleotide”.

The term “isolated polypeptide” essentially relates herein to that thepolypeptide is isolated from its natural environment. The novelglycosyltransferase polypeptide as shown in SEQ ID NO: 2 was isolatedfrom the insect Dactylopius coccus. Accordingly, as understood by theskilled person—the term “isolated polypeptide” does not cover theglycosyltransferase polypeptide of SEQ ID NO: 2 when it is naturallypresent in the genome of Dactylopius coccus. The term “isolatedpolypeptide” denotes a polypeptide preparation which contains at most10%, preferably at most 8%, more preferably at most 6%, more preferablyat most 5%, more preferably at most 4%, at most 3%, even more preferablyat most 2%, most preferably at most 1%, and even most preferably at most0.5% by weight of other polypeptide material with which it is nativelyassociated. The term “other polypeptide material with which it isnatively associated” may in relation to the novel glycosyltransferasepolypeptide as shown in SEQ ID NO: 2 be seen in relation to otherpolypeptide material with which it is natively associated in Dactylopiuscoccus. In some case—it may be preferred that the “isolated polypeptide”refers to a polypeptide which is at least 20% pure, preferably at least40% pure, more preferably at least 60% pure, even more preferably atleast 80% pure, most preferably at least 90% pure, and even mostpreferably at least 95% pure, as determined by SDS-PAGE.

The term “nucleic acid construct” as used herein refers to a nucleicacid molecule, either single- or double-stranded, which is isolated froma naturally occurring gene or which is modified to contain segments ofnucleic acids in a manner that would not otherwise exist in nature. Theterm nucleic acid construct is synonymous with the term “expressioncassette” when the nucleic acid construct contains the control sequencesrequired for expression of a coding sequence of the present invention.As known in the art control, sequences include all components, which arenecessary or advantageous for the expression of a polynucleotideencoding a polypeptide of the present invention. Each control sequencemay be native or foreign to the nucleotide sequence encoding thepolypeptide. Such control sequences include, but are not limited to, aleader, polyadenylation sequence, propeptide sequence, promoter, signalpeptide sequence, and transcription terminator. At a minimum, thecontrol sequences include a promoter, and transcriptional andtranslational stop signals. The control sequences may be provided withlinkers for the purpose of introducing specific restriction sitesfacilitating ligation of the control sequences with the coding region ofthe nucleotide sequence encoding a polypeptide.

The term “recombinant expression vector” relates to recombinantexpression vectors comprising a polynucleotide of the present invention,a promoter, and transcriptional and translational stop signals. Thevarious nucleic acids and control sequences described above may bejoined together to produce a recombinant expression vector which mayinclude one or more convenient restriction sites to allow for insertionor substitution of the nucleotide sequence encoding the polypeptide atsuch sites.

The term “recombinant host cell” should herein be understood accordingto the art. As known in the art, recombinant polynucleotide (e.g. DNA)molecules are polynucleotide (e.g. DNA) molecules formed by laboratorymethods of genetic recombination (such as molecular cloning) to bringtogether genetic material from multiple sources, creating sequences thatwould not otherwise be found in biological organisms. As understood bythe skilled person—a recombinant host cell comprises recombinantpolynucleotide (e.g. DNA) molecules and a recombinant host cell willtherefore not be understood as covering a natural wildtype cell, such ase.g. a natural wildtype Dactylopius coccus cell.

The term “Sequence Identity” relates to the relatedness between twoamino acid sequences or between two nucleotide sequences.

For purposes of the present invention, the degree of sequence identitybetween two amino acid sequences is determined using theNeedleman-Wunsch algorithm (Needleman and Wunsch, 1970, J. Mol. Biol.48: 443-453) as implemented in the Needle program of the EMBOSS package(EMBOSS: The European Molecular Biology Open Software Suite, Rice etal., 2000, Trends Genet. 16: 276-277), preferably version 3.0.0 orlater. The optional parameters used are gap open penalty of 10, gapextension penalty of 0.5, and the EBLOSUM62 (EMBOSS version of BLOSUM62)substitution matrix. The output of Needle labeled “longest identity”(obtained using the −nobrief option) is used as the percent identity andis calculated as follows:(Identical Residues×100)/(Length of Alignment−Total Number of Gaps inAlignment).For purposes of the present invention, the degree of sequence identitybetween two nucleotide sequences is determined using theNeedleman-Wunsch algorithm (Needleman and Wunsch, 1970, supra) asimplemented in the Needle program of the EMBOSS package (EMBOSS: TheEuropean Molecular Biology Open Software Suite, Rice et al., 2000,supra), preferably version 3.0.0 or later. The optional parameters usedare gap open penalty of 10, gap extension penalty of 0.5, and theEDNAFULL (EMBOSS version of NCBI NUC4.4) substitution matrix. The outputof Needle labeled “longest identity” (obtained using the −nobriefoption) is used as the percent identity and is calculated as follows:(Identical Deoxyribonucleotides×100)/(Length of Alignment−Total Numberof Gaps in Alignment).

Embodiments of the present invention are described below, by way ofexamples only.

DRAWINGS

FIG. 1: Schematic presentation of the relevant glycosyltransferaseactivity of the herein described isolated/cloned novelglycosyltransferase of SEQ ID NO:2—as illustrated in the figure it wasfound to be able to conjugate glucose to the aglycons flavokermesic acid(FK) and kermesic acid (KA).

FIG. 2: Production of glucosides of flavokermesic acid and kermesic acidusing OsCGT and SbUGT85B1. LC-MS analyses of glucosylated productsformed in assays containing UDP-glucose and flavokermesicc acid (FK) orkermesic acid (KA). Crude lysate from the E. coli strain Xjb (negativecontrol) incubated with FK (A) or KA (C). Crude lysate from Xjb cellsexpressing OsCGT incubated with FK (B) or KA (D). Crude lysate from Xjbcells expressing SbUGT85B1 incubated with FK (E) or KA (F). FK (G) andKA (H) substrates alone. The total ion chromatograms (TIC) and extractedion chromatograms for m/z 313[M-H]⁻, m/z 329[M-H]⁻, m/z 475 [M-H]⁻, m/z491 [M-H]⁻, corresponding to FK, KA, FK-monoglucoside, andKA-monoglucoside are indicated. Peak retention times are indicated inminutes.

DETAILED DESCRIPTION OF THE INVENTION

The present application includes a Sequence Listing which has beensubmitted in ASCII computer readable format (CFR) and in paper format,both via EFS-Web, and are hereby incorporated by reference in theirentirety.

A Novel Isolated Glycosyltransferase Polypeptide as Described Herein

When there herein is referred to an isolated glycosyltransferasepolypeptide as described herein there is referred to an isolatedglycosyltransferase polypeptide of the first aspect of the inventionand/or herein relevant embodiments thereof.

As discussed above—the term “isolated polypeptide” essentially relatesto that the polypeptide is isolated from its natural environment. Theherein described novel glycosyltransferase polypeptide as shown in SEQID NO: 2 was isolated from the insect Dactylopius coccus. Accordingly,as understood by the skilled person in the present context—the term“isolated polypeptide” does not cover the glycosyltransferasepolypeptide of SEQ ID NO: 2 when it is naturally present in the genomeof Dactylopius coccus.

Preferably, the isolated glycosyltransferase polypeptide as describedherein denotes a polypeptide preparation which contains at most 10%,preferably at most 8%, more preferably at most 6%, more preferably atmost 5%, more preferably at most 4%, at most 3%, even more preferably atmost 2%, most preferably at most 1%, and even most preferably at most0.5% by weight of other polypeptide material with which it is nativelyassociated.

As understood by the skilled person, the term “other polypeptidematerial with which it is natively associated” may in relation to thenovel glycosyltransferase polypeptide as shown in SEQ ID NO: 2 be seenas relation to other polypeptide material with which it is nativelyassociated in Dactylopius coccus.

In some case—it may be preferred that the isolated glycosyltransferasepolypeptide as described herein refers to a polypeptide which is atleast 20% pure, preferably at least 40% pure, more preferably at least60% pure, even more preferably at least 80% pure, most preferably atleast 90% pure, and even most preferably at least 95% pure, asdetermined by SDS-PAGE.

Based on e.g. the sequence information disclosed herein—it is routinework for the skilled person to obtain an isolated glycosyltransferasepolypeptide as described herein.

This may e.g. be done by recombinant expression in a suitablerecombinant host cell according to procedures known in the art.

Accordingly, it is not believed necessary to describe such standardknown recombinant expression procedures in many details herein.

Preferably, the isolated glycosyltransferase polypeptide as describedherein is capable of:

-   -   (I): conjugating glucose to flavokermesic acid (FK); and    -   (II): conjugating glucose to kermesic acid (KA).

A preferred embodiment relates to wherein the glycosyltransferasepolypeptide of the first aspect is:

-   -   (a) a polypeptide comprising an amino acid sequence which has at        least 80% identity with amino acids 1 to 515 of SEQ ID NO:2;        more preferably    -   (a) a polypeptide comprising an amino acid sequence which has at        least 90% identity with amino acids 1 to 515 of SEQ ID NO:2;        even more preferably    -   (a) a polypeptide comprising an amino acid sequence which has at        least 95% identity with amino acids 1 to 515 of SEQ ID NO:2; and        most preferably    -   (a) a polypeptide comprising an amino acid sequence which has at        least 98% identity with amino acids 1 to 515 of SEQ ID NO:2.

It may be preferred that the glycosyltransferase polypeptide of thefirst aspect is a polypeptide comprising an amino acid sequence withamino acids 1 to 515 of SEQ ID NO:2.

A preferred embodiment relates to wherein the glycosyltransferasepolypeptide of the first aspect is:

-   -   (b) a polypeptide comprising an amino acid sequence which has at        least 80% identity with amino acids 20 to 468 of SEQ ID NO:2;        more preferably    -   (b) a polypeptide comprising an amino acid sequence which has at        least 90% identity with amino acids 20 to 468 of SEQ ID NO:2;        even more preferably    -   (b) a polypeptide comprising an amino acid sequence which has at        least 95% identity with amino acids 20 to 468 of SEQ ID NO:2;        and most preferably    -   (b) a polypeptide comprising an amino acid sequence which has at        least 98% identity with amino acids 20 to 468 of SEQ ID NO:2.

It may be preferred that the glycosyltransferase polypeptide of thefirst aspect is a polypeptide comprising an amino acid sequence withamino acids 20 to 468 of SEQ ID NO:2.

A preferred embodiment relates to wherein the glycosyltransferasepolypeptide of the first aspect is:

-   -   (c) a polypeptide which is encoded by a polynucleotide which        hybridizes under at least medium-high stringency conditions        with (i) nucleotides 1 to 1548 of SEQ ID NO:1 or (ii) a        complementary strand of (i); more preferably    -   (c) a polypeptide which is encoded by a polynucleotide which        hybridizes under at least high stringency conditions with (i)        nucleotides 1 to 1548 of SEQ ID NO:1 or (ii) a complementary        strand of (i); and even more preferably    -   (c) a polypeptide which is encoded by a polynucleotide which        hybridizes under at least very stringency conditions with (i)        nucleotides 1 to 1548 of SEQ ID NO:1 or (ii) a complementary        strand of (i).

It is routine work for the skilled person to make a variant of anisolated glycosyltransferase polypeptide as described herein—i.e. avariant, wherein e.g. one or more amino acids of e.g. SEQ ID NO:2 havebeen modified/altered.

Further—as known to the skilled person if such variant changes are nottoo drastic it will be plausible that the enzyme would maintain itsrelevant GT activity.

A preferred embodiment relates to wherein the glycosyltransferasepolypeptide of the first aspect is:

(a) a polypeptide comprising an amino acid sequence with amino acids 1to 515 of SEQ ID NO:2 or a variant thereof, wherein the variantcomprises an alteration at one or more (several) positions of SEQ IDNO:2 and wherein the variant comprises less than 50 alterations, morepreferably less than 40 alterations, even more preferably less than 20alterations and most preferably less than 10 alterations.

In a preferred embodiment the term “an alteration at one or more(several) positions of SEQ ID NO:2” refers to 1 to 10 alterations in SEQID NO:2.

According to the art—the term “variant” means herein a peptide havingthe relevant GT activity comprising an alteration, i.e., a substitution,insertion, and/or deletion, at one or more (several) positions. Asubstitution means a replacement of an amino acid occupying a positionwith a different amino acid; a deletion means removal of an amino acidoccupying a position; and an insertion means adding 1-3 amino acidsadjacent to an amino acid occupying a position.

The amino acid may be natural or unnatural amino acids—for instance,substitution with e.g. a particularly D-isomers (or D-forms) of e.g.D-alanine could theoretically be possible.

In a preferred embodiment the glycosyltransferase polypeptide of thefirst aspect is a GT which is membrane bound or insoluble in water.

Isolated Polynucleotide Comprising a Nucleotide Sequence which Encodesthe Glycosytransferase Polypeptide as Described Herein

As discussed above—a second aspect of the present invention relates toan isolated polynucleotide comprising a nucleotide sequence whichencodes the polypeptide of the first aspect and/or herein relevantembodiments thereof.

The term “isolated polynucleotide” may herein alternatively be termed“cloned polynucleotide”.

As discussed above—the term “isolated polynucleotide” essentiallyrelates to that the polynucleotide is isolated from its naturalenvironment—said in other words that the polynucleotide preparation isessentially free of other polynucleotide material with which it isnatively associated. The polynucleotide sequence encoding the describedisolated/cloned novel glycosyltransferase is shown in SEQ ID NO: 1 andit was isolated from the insect Dactylopius coccus. Accordingly, asunderstood by the skilled person—the term isolated polynucleotide doesnot cover the polynucleotide of SEQ ID NO: 1 when it is naturallypresent in the genome of Dactylopius coccus.

The term “isolated polynucleotide” essentially relates to that theisolated polynucleotide is in a form suitable for use within geneticallyengineered protein production systems.

Thus, an isolated polynucleotide contains at most 10%, preferably atmost 8%, more preferably at most 6%, more preferably at most 5%, morepreferably at most 4%, more preferably at most 3%, even more preferablyat most 2%, most preferably at most 1%, and even most preferably at most0.5% by weight of other polynucleotide material with which it isnatively associated.

Based on e.g. the sequence information disclosed herein—it is routinework for the skilled person to obtain an isolated polynucleotide asdescribed herein.

This may e.g. be done by recombinant expression in a suitablerecombinant host cell according to procedures known in the art.

Accordingly, it is not believed necessary to describe such standardknown recombinant expression procedures in many details herein.

A Nucleic Acid Construct Comprising the Isolated Polynucleotide asDescribed Herein

As discussed above—a third aspect of the present invention relates to anucleic acid construct comprising the isolated polynucleotide of thesecond aspect and/or herein relevant embodiments thereof operably linkedto one or more control sequences that direct the production of thepolypeptide in an expression host.

According to the art—the term “nucleic acid construct” as used hereinrefers to a nucleic acid molecule, either single- or double-stranded,which is isolated from a naturally occurring gene or which is modifiedto contain segments of nucleic acids in a manner that would nototherwise exist in nature.

The term nucleic acid construct is synonymous with the term “expressioncassette” when the nucleic acid construct contains the control sequencesrequired for expression of a coding sequence of the present invention.As known in the art control sequences include all components, which arenecessary or advantageous for the expression of a polynucleotideencoding a polypeptide of the present invention.

Based on e.g. the sequence information disclosed herein—it is routinework for the skilled person to make a relevant nucleic acidconstruct—for instance, based on the prior art the skilled person knowsnumerous different suitable control sequences for the expression of apolynucleotide encoding a polypeptide of the present invention.Accordingly, it is not believed necessary to describe such standardknown technical elements in many details herein.

A Recombinant Expression Vector Comprising the Nucleic Acid Construct asDescribed Herein

As discussed above—a fourth aspect of the present invention relates to arecombinant expression vector comprising the nucleic acid construct ofthe third aspect and/or herein relevant embodiments thereof.

According to the art—the term “recombinant expression vector” relates torecombinant expression vectors comprising a polynucleotide of thepresent invention, a promoter, and transcriptional and translationalstop signals. The various nucleic acids and control sequences describedabove may be joined together to produce a recombinant expression vectorwhich may include one or more convenient restriction sites to allow forinsertion or substitution of the nucleotide sequence encoding thepolypeptide at such sites.

Based on e.g. the sequence information disclosed herein—it is routinework for the skilled person to make a relevant recombinant expressionvector—for instance, based on the prior art the skilled person knowsnumerous different suitable promoter, and transcriptional andtranslational stop signals.

Accordingly, it is not believed necessary to describe such standardknown technical elements in many details herein.

A Recombinant Host Cell Comprising the Nucleic Acid Construct asDescribed Herein

As discussed above—a fifth aspect of the present invention relates to arecombinant host cell comprising the nucleic acid construct of the thirdaspect and/or herein relevant embodiments thereof.

The term “recombinant host cell” should herein be understood accordingto the art. As known in the art, recombinant polynucleotide (e.g. DNA)molecules are polynucleotide (e.g. DNA) molecules formed by laboratorymethods of genetic recombination (such as molecular cloning) to bringtogether genetic material from multiple sources, creating sequences thatwould not otherwise be found in biological organisms. As understood bythe skilled person—a recombinant host cell comprises recombinantpolynucleotide (e.g. DNA) molecules and a recombinant host cell willtherefore not be understood as covering a natural wildtype cell, such ase.g. a natural wildtype Dactylopius coccus cell.

Based on e.g. the sequence information disclosed herein—it is routinework for the skilled person to make a relevant recombinant host cell—forinstance, based on the prior art the skilled person knows numerousdifferent suitable recombinant host cells that for years have been usedas recombinant host cells for e.g. expression of different polypeptidesof interest.

The recombinant host cell may be any suitable cell such as anyeukaryotic cell [e.g. mammalian cells (such as e.g. Chinese hamsterovary (CHO) cells) or a plant cell] or any prokaryotic cell.

Particularly preferred is wherein the recombinant host cell is a plantcell producing flavokermesic acid/kermesic acid or other relatedcompound such as e.g. rhubarb plant cell.

Preferably the recombinant host cell is a cell selected from the groupconsisting of a filamentous fungal cell and a microorganism cell.

Filamentous fungi include all filamentous forms of the subdivisionEumycota and Oomycota (as defined by Hawksworth et al., 1995, supra).The filamentous fungi are characterized by a vegetative myceliumcomposed of chitin, cellulose, glucan, chitosan, mannan, and othercomplex polysaccharides. Vegetative growth is by hyphal elongation andcarbon catabolism is obligately aerobic. In contrast, vegetative growthby yeasts such as Saccharomyces cerevisiae is by budding of aunicellular thallus and carbon catabolism may be fermentative.

It may be preferred that the filamentous fungal cell is a cell of aspecies of, but not limited to, Acremonium, Aspergillus, Fusarium,Humicola, Mucor, Myceliophthora, Neurospora, Penicillium, Thielavia,Tolypocladium, and Trichoderma or a teleomorph or synonym thereof.

A preferred Aspergillus cell is Aspergillus niger or Aspergillus oryzae.

A preferred microorganism cell herein is a microorganism cell selectedfrom the group consisting of a yeast cell and prokaryotic cell.

A preferred yeast cell is a yeast cell selected from the groupconsisting of Ascomycetes, Basidiomycetes and fungi imperfecti.Preferably a yeast cell selected from the group consisting ofAscomycetes.

Preferred Ascomycetes yeast cell selected from the group consisting ofAshbya, Botryoascus, Debaryomyces, Hansenula, Kluveromyces, Lipomyces,Saccharomyces spp e.g. Saccharomyces cerevisiae, Pichia spp.,Schizosaccharomyces spp.

A preferred yeast cell is a yeast cell selected from the groupconsisting of Saccharomyces spp, e.g. Saccharomyces cerevisiae, andPichia spp.

A preferred prokaryotic cell is selected from the group consisting ofBacillus, Streptomyces, Corynebacterium, Pseudomonas, lactic acidbacteria and an E. coli cell.

A preferred Bacillus cell is B. subtilis, B. amyloliquefaciens or B.licheniformis.

A preferred Streptomyces cell is S. setonii or S. coelicolor.

A preferred Corynebacterium cell is C. glutamicum.

A preferred Pseudomonas cell is P. putida or P. fluorescens.

A Method for Producing Flavokermesic Acid (FK) Glycoside and/or KemesicAcid (KA)

As discussed above—a sixth aspect of the present invention relates to amethod for producing flavokermesic acid (FK) glycoside and/or kermesicacid (KA) glycoside, wherein the method comprises following steps:

(A): contacting in vitro or in vivo in a recombinant host cellcomprising a glycosyltransferase gene encoding a glycosyltransferase:

-   -   (a1): flavokermesic acid (FK) with a glycosyltransferase capable        of glycosylating the flavokermesic acid under suitable        conditions wherein there is produced the flavokermesic acid        glycoside; and/or    -   (a2): kermesic acid (KA) with a glycosyltransferase capable of        glycosylating the kermesic acid under suitable conditions        wherein there is produced the kermesic acid glycoside.

It may be preferred that the recombinant host cell in step (A) is arecombinant host cell comprising a recombinant glycosyltransferase geneencoding a glycosyltransferase.

Preferably, the glycosyltransferase in step (a2) is aglucosyltransferase and there thereby in step (a2) is produced kermesicacid glucoside, preferably wherein the produced kermesic acid glucosideis Carminic acid (FIG. 1 herein shows the structure of Carminic acid).

It may be preferred that the glycosyltransferase in step (a1) is aglucosyltransferase and there thereby in step (a1) is producedflavokermesic acid glucoside, preferably wherein the producedflavokermesic acid glucoside is the compound DcII (FIG. 1 herein showsthe structure of the compound DcII).

When the produced compound in step (a1) is DcII it may be preferred touse this DcII as an intermediate to make Carminic acid.

This may be done by chemical synthesis and the skilled person knowsbased on his common general knowledge how to do this.

Alternatively, it may be done enzymatically by e.g. using a suitableoxygenase. An example of a suitable oxygenase is cytochrome P450superfamily of monooxygenases (officially abbreviated as CYP) enzyme.Other examples are flavine monooxygenases or different types ofdioxygenases, this list not to be considered excluding the involvementof other classes of enzymes

As known in the art—the most common reaction catalyzed by cytochromesP450 is a monooxygenase reaction, e.g., insertion of one atom of oxygeninto a substrate.

As understood by the skilled person in the present context—the termsflavokermesic acid (FK) and/or kermesic acid (KA) aglycons of step (a)of the method of the sixth aspect as discussed herein should beunderstood as the FK and/or KA specific compounds shown in FIG. 1 andequivalent analogs of these specific compounds with minor substituents(e.g. a FK methyl ester).

As understood in by the skilled person—if FK methyl ester is used asaglycon in step (a) of the method of the sixth aspect then there willvia the glycosylation step be generated a FK methyl ester glycoside,which by routine removal of the methyl group will generateDcII—accordingly FK methyl ester aglycon may be seen as equivalent to FKaglycon in relation to the method of the sixth aspect as discussedherein.

In step (a) of the method of the sixth aspect is specified that there isused a glycosyltransferase capable of glycosylating FK and/orKA—accordingly it is understood that the GT must be capable of doingthis.

It may be preferred to purify the glycoside produced in step (A)—i.e. instep (a1) and/or in step (a2).

Accordingly it may be preferred that the method of the sixth aspectcomprises a further step (B) with following steps:

-   -   (B): purifying the produced glycoside in step (a1) and/or in        step (a2) whereby one gets a composition, wherein at least 5%        w/w (preferably at least 10% w/w, more preferably at least 50%        w/w and most preferably at least 80% w/w) of the compounds in        the composition is the produced flavokermesic acid glycoside        and/or kermesic acid glycoside.

The skilled person knows how to purify such glycoside compounds and itmay be done according to the art.

The purifying step (B) may be particularly preferred when:

-   -   the produced glycoside in step (a2) is Carminic acid;    -   the produced glycoside in step (a1) is compound DcII; and/or    -   the produced glycoside in step (a1) is compound DcII and it is        used as an intermediate to make Carminic acid.

As discussed herein—in working Examples there was made a contacting invitro of flavokermesic acid (FK) and/or kermesic acid (KA) with theglycosyltransferase of SEQ ID NO:2. It may be seen as routine work forthe skilled person to perform such an in vitro contacting step.

The glycosyltransferase of SEQ ID NO:2 was recombinantly expressed in ayeast cell (see working Example herein)—accordingly, in a workingExample herein there was made a recombinant yeast host cell comprising arecombinant glycosyltransferase gene encoding a glycosyltransferase ofSEQ ID NO:2.

It is believed that if flavokermesic acid (FK) and/or kermesic acid (KA)would be added under suitable condition to a fermentation medium the FKand/or KA compound(s) would enter into e.g. yeast cells fermented in themedium—accordingly, if e.g. the yeast cells are recombinant yeast hostcells comprising a recombinant glycosyltransferase gene encoding aglycosyltransferase then there would be made a contacting in vivo in arecombinant host cell of FK and/or KA with a glycosyltransferase.

In a preferred embodiment the contacting in step (A) is in vivo and therecombinant host cell is a yeast cell, preferably wherein the yeast cellis selected from the group consisting of Saccharomyces spp (e.g.Saccharomyces cerevisiae) and Pichia spp.

Above is described preferred recombinant host cells—these preferredrecombinant host cells may also be preferred recombinant host cells inrelation to the method of the sixth aspect of the present invention.

In the present context—it may be said that it is within the skilledperson's common knowledge to identify a suitable recombinant host cellto perform the in vivo contacting step (A) of the method of the sixthaspect and it is not believed that it is necessary to describe this inmany details herein.

Above is discussed that preferred recombinant host cells may e.g. be amicroorganism cell or a filamentous fungal cell—these cells may bepreferred recombinant host cells in relation to the method of the sixthaspect.

It may be possible to make a recombinant host cell (e.g. a recombinanthost microorganism cell) which comprises a gene encoding a productinvolved in the biosynthesis pathway leading to flavokermesic acid (FK)and/or kermesic acid (KA) and such a recombinant host cell could bepreferred herein.

Accordingly, it may be preferred that the contacting in step (A) iscontacting in vivo in a recombinant host cell comprising a recombinantglycosyltransferase gene encoding a glycosyltransferase and a geneencoding a product involved in the biosynthesis pathway leading toflavokermesic acid (FK) and/or kermesic acid (KA).

As discussed in working Example herein—the GT of SEQ ID NO:2 is membranebound or hydrophobic/insoluble in vivo and in water. When productioncells or fractions of cells containing the membrane bound GT areseparated from the product (e.g. carminic acid), the GT can essentiallynot be present in the fraction where the more solubleproduct/hydrophilic product is present. This is an advantage forobtaining a final product (e.g. carminic acid product/composition) whichis essentially totally free of the recombinant GT.

Because the substrates glycosylated by the GT may be hydrophobicaglycons, the aglycons would be expected to partly accumulate inmembranes and other hydrophobic parts of the production cells. By theuse of a membrane bound GT a more efficient glycosylation of hydrophobiccompounds present in e.g. membranes is obtained

Accordingly, in a preferred embodiment the glycosyltransferase used inthe method of the sixth aspect is a GT which is membrane bound orinsoluble in water.

In a preferred embodiment—the glycosyltransferase in step (A) of themethod of the sixth aspect is a glycosyltransferase of the first aspectand/or herein relevant embodiments thereof.

As discussed herein—the identified data/results of working Examples 4show that herein relevant GT enzymes can be identified in e.g. Sorghumand rice plants.

The Sorghum polypeptide sequence (Genbank ID number: AAF17077.1) isshown as SEQ ID NO: 4 herein.

The rice polypeptide sequence (Genbank ID number: CAQ77160.1) is shownas SEQ ID NO: 5 herein.

It may be relevant that the glycosyltransferase in step (A) of themethod of the sixth aspect is a glycosyltransferase comprising an aminoacid sequence which has at least 70% (preferably at least 80%, morepreferably at least 90% and even more preferably at least 98%) identitywith amino acids 1 to 492 of SEQ ID NO:4.

It may be relevant that the glycosyltransferase in step (A) of themethod of the sixth aspect is a glycosyltransferase comprising an aminoacid sequence which has at least 70% (preferably at least 80%, morepreferably at least 90% and even more preferably at least 98%) identitywith amino acids 1 to 471 of SEQ ID NO:5.

EXAMPLES Example 1—Cloning of D. coccus GT and Test of its FK and KAActivity

Materials and Methods

Purification of DNA and mRNA

Fresh frozen Dactylopius coccus (were obtained from Lanzarote). Freshfrozen Porphyrophora polonica were obtained from Poland. The frozeninsects were ground into powder under liquid nitrogen and DNA/RNA waspurified: DNA was purified using DNeasy Blood & Tissue kit (Qiagen),according to the protocol of the supplier. RNA was purified using RNeasymini kit (Qiagen) according to the protocol of the supplier. EucaryotemRNA was made into cDNA using RT² Easy First Strand Kit (Qiagen)according to the protocol of the supplier using poly-dT priming of therevers transcriptase reaction.

Sequencing of DNA and RNA:

DNA and cDNA were sent for sequencing at BGI (Shenzen, China) forsequencing using 100 bp paired-end Illumina technology according to theprotocol of Illumina at a coverage of approximately 60-100× and theoutput in fastq data format.

Analysis of DNA and RNA/cDNA Sequences:

The obtained fastq-sequences of DNA and RNA/cDNA were imported intoGenomic Workbench version 5.4 (CLC-bio, Århus, Denmark) and assembledusing the de novo assembling algorithm into contigs. Output files wereexported as FASTA format. RNA/cDNA FASTA files were then imported intoIOGMA v. 10 (Genostar, Grenoble, France) and putitative genes wereidentified using the “hidden Markov-Matrix-based prokaryote gene-finder.

Putative genes were annotated using BLAST (basic local alignmentsequence tool) against genbank (NCBI) using as well the nucleotidesequence as the translated protein sequence. The putative genes werealso annotated by similarity comparison to PFAM databases of proteinfamilies.

Preparation of Protein Fractions from D. coccus

Three grams of fresh D. coccus insects were homogenized in 120 mL ofisolation buffer [350 mM sucrose, 20 mM Tricine (pH 7.9), 10 mM NaCl, 5mM DTT, 1 mM PMSF) containing 0.3 g polyvinylpolypyrrolidone. Thehomogenate was filtered through a nylon cloth (22 μm mesh) andcentrifuged for (10 min, 10,000×g at 4° C.). The supernatant wascentrifuged (1 h, 105,000×g, at 4° C.), yielding a soluble and amembrane bound protein fraction. The soluble protein fraction wasconcentrated to 1 mL and buffer-exchanged with 20 mM Tricine (pH 7.9), 5mM DTT by using Amicon ultrafugation-3K devices (Millipore). Themembrane bound protein pellet was washed 3 times by resuspending thepellet in 60 mL of 20 mM Tricine (pH 7.9), 5 mM DTT using a martenpaintbrush followed by re-centrifugation. The membrane bound proteinpellet was finally resuspended in 1 mL 20 mM Tricine (pH 7.9), 5 mM DTT.The soluble protein fraction and the membrane bound protein fractionwere analyzed for glycosylation activity.

Purification of a Flavokermesic Acid/Kermesic Acid-Specific GT Activityfrom D. coccus Membrane Proteins

A membrane bound protein fraction isolated from 3 g fresh D. coccusinsects was solubilized by adding 1% (v/v) Triton x-100 (reduced form)and gently stirring for 1.5 h in the cold. The Triton x-100 treatedsolution was centrifuged (1 h, 105,000×g, at 4° C.) and the supernatantwas isolated and applied to a column packed with 2 mL Q-sepharose Fastflow (Pharmacia). The column was washed in 4 mL of buffer A [20 mMTricine (pH 7.9), 0.1% (v/v) Triton x-100 (reduced form), 50 mM NaCl]and proteins were eluted with 20 mM Tricine (pH 7.9), 0.1% (v/v) Tritonx-100 (reduced form)] using a discontinuous NaCl gradient from 100mM-500 mM, (with 50 mM increments). 0.5-ml-fractions were collected,desalted, analyzed by SDS-PAGE and monitored for glucosylation activityusing the described radiolabeled glucosylation enzyme assay. A fractioncontaining enriched flavokermesic acid/kermesic acid-specific GTactivity was subjected to peptide mass fingerprinting analysis.

Enzyme Assays and Glucoside Product Detection

Glucosylation of flavokermesic acid and kermesic acid was performed inassay mixtures of 60 μL containing 20 mM Tricine (pH 7.9), 3.3 μmUDP[14C]glucose and 20 uL protein extract (membrane bound or solubleprotein). Reactions were incubated for 0.5 h at 30° C. and terminated byadding 180 μL of methanol. Samples were centrifuged at 16,000×g for 5min at 4° C. and supernatant was spotted on TLC plates (silica gel 60F254 plates; Merck). Assay products were resolved indichloromethane:methanol:formic acid (7:2:2, by volume). Radiolabeledproducts were visualized using a STORM 840 PhosphorImager (MolecularDynamics, http://www.moleculardynamics.com).

Expression of Codon Optimized DcUGT2, DcUGT4 and DcUGT5 in S. cerevisiae

A synthetic codon optimized version of DcUGT2 and two other putative GTsequences from the D. coccus transcriptome termed DcUGT4 and DcUGT5 foryeast expression was purchased from GenScript with flanking gatewayrecombination attL sites. The synthetic fragments were used as PCRtemplates with specific primers to generate the corresponding C-terminalStrepII-tagged versions. The six gene constructs (tagged and non-taggedfragments) were cloned into the gateway destination plasmid pYES-DEST52(Invitrogen) using LR clonasell enzyme mix. The six pYES-DEST52 plasmidconstructs were transformed separately into the Invsc1 yeast strain(Invitrogen) and positive transformants were verified by PCR.Heterologous protein production was performed according to theinstructions of the pYES-DEST52 gateway vector (Invitrogen). Productionof heterologous StrepII-tagged protein was verified by western blottingusing anti-Strep antibody. A membrane bound protein fraction wasprepared from verified yeast transformants as described in (D. Pompon,B. Louerat, A. Bronine, P. Urban, Yeast expression of animal and plantP450s in optimized redox environments, Methods Enzymol. 272 (1996)51-64) and screened for glucosylation activity towards flavokermesicacid/kermesic acid. The yeast optimized sequence is shown in SEQ ID NO:3 herein.

LC-MS-MS

LC/MS was performed on an Agilent Q-TOF with the following HPLC system:Column Kinetix 2.6μ XB-C18 100A (100×4.60 mm, Phenomenex). Solvent A(900 ml deionized water, 100 ml methanol and 50 ml formic acid). SolventB (700 ml methanol, 300 ml deionized water and 50 ml formic acid).

Flow 0.8 ml/min. 35° C.

Gradient elution. 0-1 min 100% A. Linear gradient to 83% A 3 min. lineargradient to 63% A 6 min, linear gradient to 45% A 9 min, linear gradientto 27% A 12 min, linear gradient to 10% A 15 min, linear gradient to 3%A 17 min, linear gradient to 2% A 19 min, linear gradient to 0% A 20min, 0% A 22 min, linear gradient to 100% A 25 min. Retention times were7.6 min for carminic acid, 7.8 min for DC II, 13.7 min for flavokermesicacid and 13.9 min for kermesic acid.

Results:

The ability to glycosylate flavokermesic acid/kermesic acid usingC14-UDP-glucose as a substrate was detected in homogenized D. coccusinsects. The activity was shown to be membrane bound and the activitywas purified and the purified proteins were submitted to proteomicsanalysis. It was shown that the enzymatic activity was to come from apolypeptide with a sequence corresponding to our candidate gene DcUGT2.

As discussed above—the herein relevant glycosyltransferase enzyme of SEQID NO: 2 may herein be termed “DcUGT2”.

The amino acid sequence of DcUGT2 shows less than 45% homology to anyknown glycosyl transferase.

Knowing that cloning the wildtype sequence into yeast had given norelevant enzyme activity, we redesigned the nucleotide sequence ofDcUGT2 to a sequence coding for the same polypeptide but usingnucleotide codons optimized for S. cerevisiae, a process called codonoptimization (the S. cerevisiae optimized sequence is shown as SEQ IDNo. 3 herein). Subsequently the codon optimized sequence of DcUGT2 wascloned and expressed in yeast. The heterologous yeast strain contains amembrane bound enzyme activity capable of glucosylating kermesic acidand flavokermesic acid. After obtaining peptide mass fingerprinting datafrom a D. coccus protein fraction enriched with GT activity towardsflavokermesic acid/kermesic acid, we matched the peptide masses to thetranscriptomic dataset and identified three putative UGTs (DcUGT2,DcUGT4 and DcUGT5).

Heterologous expression of the three candidates in yeast revealed thatonly one of these UGTs, namely DcUGT2 was responsible for the observedglucosylation activity towards flavokermesic acid/kermesic acid in theD. coccus protein fraction.

A viscozyme treatment of the generated C-14 radiolabelled glucoside,showed that it was resistant towards hydrolysis, further suggesting thatthe DcUGT2 is a C-GT, responsible for producing DCII and carminic acid.

A LC-MS-MS showed formation of products with the same retention time,spectrum, molecular mass and molecular degradation pattern as DcII andcarminic acid respectively.

Conclusion

The result of this example 1 demonstrated that it was not an easy taskto isolate/clone the herein relevant glycosyltransferase enzyme of SEQID NO: 2, which may herein be termed “DcUGT2”.

For instance, the identified gene sequences of the genome andtranscriptome of D. coccus insects were analyzed for similarity toherein relevant public known C-glycosyltransferase sequences and theresult was negative in the sense that none of the identified genesequences of the genome/transcriptome showed herein significantsimilarity to publicly known herein relevant C-glycosyltransferasesequences.

However, even though the bioinformatic sequence similarity analysiscould be said to indicate that the genome of Dactylopius coccus wouldnot comprise a gene encoding a herein relevant glycosyltransferase—thepresent inventors continued to investigate the matter and the presentinventors identified a Dactylopius coccus extract (including extracts ofthe endosymbionts present in D. coccus) with herein relevant GT activityand by a combination of herein relevant purification and testing stepsthe inventors were finally able to get a relatively purefraction/composition wherefrom it was possible to obtain several partialamino acid sequences of possible GT enzyme candidates.

The present inventors tested the activity of the herein describedisolated/cloned novel glycosyltransferase of SEQ ID NO: 2 (DcUGT2) andfound that it was able to conjugate glucose to the aglyconsflavokermesic acid (FK) and kermesic acid (KA)—see FIG. 1 herein.

Example 2—Testing KA GT Activity of Prior Art Known UrdGT2

As discussed above—the UrdGT2 is described in the article Baig et al(Angew Chem Int Ed Engl. 2006 Nov. 27; 45(46):7842-6).

As discussed above—this article describes that UrdGT2 is capable ofglycosylating different aglycon molecules that may be consideredstructurally similar to the herein relevant Kermesic acid (KA) andFlavokermesic acid (FK) aglycons.

A codon optimized synthetic version of UrdGT2 for E. coli expression wascloned and recombinantly expressed in E. coli. A crude soluble proteinextract containing the recombinant UrdGT2 was obtained—i.e. an extractcomprising the UrdGT2

The UrdGT2 GT activity was analyzed in vitro using either UDP-glucose orTDP-glucose as a sugar donor and FA/KA as aglycone substrates. Noactivity was detected towards these aglycons—i.e. no herein relevant GTactivity was identified in relation to these aglycons.

However, it was confirmed that the recombinant UrdGT2 was active, asdemonstrated by the in vitro formation of a C14-radiolabelled glucosidederived from the glucosylation of an unidentified compound in the crudeE. coli extract.

Example 3—GT Activity in Aloe Plant and Haworthia Plant

Isolation and Test of GT Activity from Aloe

-   -   1) The plant was washed from soil particles and separated        into: A) Root, B) Green leaf tissue and C) the gel material from        the leaf    -   2) 5 g of tissue was frozen immediately in liquid nitrogen and        ground in a cold mortar with a pestle to a fine powder.    -   3) 20 mL of cold extraction buffer [20 mM Tricine-HCl, 10 mM        NaCl, 5 mM DTT, 1 mM PMSF, pH 7.9] containing a Complete        protease inhibitor without EDTA (Roche), 0.1% (w/v) proteamine        sulfate and 0.5 g of PVPP were added to the powder and vortexed.    -   4) The homogenate was gently stirred at 4° C. for 10 min and        then centrifuged at 12,000×g at 4° C. for 5 min.    -   5) Supernatant was isolated and 1 mL of 2% (w/v) proteamine        sulfate in 20 mM Tricine-HCl, pH 7.9 was added dropwise over 2        min at 4° C. under constant stirring.    -   6) The supernatant was filtered through 2 pieces of nylon mesh.        The filtered supernatant was then centrifuged at 12,000×g at        4° C. for 5 min.    -   7) The supernatant was isolated and ultracentrifuged at        110,000×g at 4° C. for 1 h.    -   8) The soluble protein fraction (supernatant) was isolated and        buffer-exchanged 5 times with 20 mM Tricine-HCl, pH 7.9        containing 5 mM DTT using a Amicon Ultra centrifugal filter        device-3K (Millipore)    -   9) 20 μL soluble protein extract was incubated in a total        reaction volume of 60 μL containing UDP-glucose (1.25 mM final        conc.) and either FK (50 M final conc.), KA (50 μM final conc)        or MeO-FK/EtO-FK (50 μM/50 μM final conc) for 2 h at 30° C.,        shaking at 650 rpm.    -   10) Enzyme reactions were terminated with 180 μL cold methanol        and filtered through a 0.45 micron filter and subjected to        HPLC-MS analysis.

TABLE 1 Glucosides formed in in vitro glucosylation assays using enzymeextracts from Aloe. m/z [M − H]⁻ values 503 m/z Aloe 475 m/z 491 m/z 489m/z [M − H]⁻ [M − H]⁻ Soluble [M − H]⁻ [M − H]⁻ MeOFK- EtOFK- proteinFK-monoglc KA-monoglc monoglc monoglc Leaf 3.73 3.71 5.81 6.63 Gel Root3.71

Crude soluble enzyme extracts of three Aloe tissues, green leaf material(Leaf), gel material from the leaf (Gel) and Root were tested forglucosylation activity towards flavokermesic acid (FK), kermesic acid(KA), methyl ester of flavokermesic acid (MeOFK) and ethyl ester offlavokermesic acid (EtOFK). Numbers correspond to retention times (min)after HPLC-MS separation of the novel glucosides formed in vitro (Table1).

The m/z values 475 and 491 are the same m/z values as are obtained forDcII and CA, respectively, solubilized in similar solutions. Both m/zvalues are 162 (m/z value of glucose in a glucoside) higher than the m/zvalues of the FK and KA indicating that the glucose moiety fromUDP-glucose in the reaction buffer has been transferred to the aglyconeby a GT in the extract. The m/z [M-H] values 489 and 503 are also 162higher than the m/z values obtained with MeOFK and EtOFK, respectively,indicating that a glucose unit has been added to both MeOFK and EtOFK bya GT present in the extract.

Isolation and Test of GT Activity from Haworthia limifolia

The procedure was as described for Aloe but plant tissue analyzed werefollowing: A) Green leaf tissue, B) Gel material from the leaf, C) Basetissue (pink part between root and stem) and D) Root tissue.

Crude soluble enzyme extracts of four Haworthia limifolia tissues, greenleaf material (Leaf), gel material from the leaf (Gel), pink tissuebetween root and stem (Base) and Root were tested for glucosylationactivity towards flavokermesic acid (FK), kermesic acid (KA), methylester of flavokermesic acid (MeOFK) and ethyl ester of flavokermesicacid (EtOFK). Numbers correspond to retention times (min) after HPLC-MSseparation of the novel glucosides formed in vitro (Table 2).

TABLE 2 Glucosides formed in in vitro glucosylation assays using enzymeextracts from Haworthia limifolia. m/z [M − H]⁻ values 503 m/z Haworthia475 m/z 491 m/z 489 m/z [M − H]⁻ [M − H]⁻ Soluble [M − H]⁻ [M − H]⁻MeOFK- EtOFK- protein FK-monoglc KA-monoglc monoglc monoglc Leaf 3.733.71 5.81 6.63 Gel Base 3.73 3.71 5.81 6.63 Root 3.73 3.71 5.81 6.63

The m/z values 475 and 491 are the same m/z values as are obtained forDcII and CA, respectively, solubilized in similar solutions. Both m/zvalues are 162 (m/z value of glucose in a glucoside) higher than the m/zvalues of the FK and KA indicating that the glucose moiety fromUDP-glucose in the reaction buffer has been transferred to the aglyconeby a GT in the extract. The m/z [M-H] values 489 and 503 are also 162higher than the m/z values obtained with MeOFK and EtOFK, respectively,indicating that a glucose unit has been added to both MeOFK and EtOFK bya GT present in the extract.

Conclusion

The results of this example demonstrate that herein relevantglycosyltransferase (GT) enzymes can be identified in Aloe plants andHaworthia plants.

Said in other words, Aloe plants and Haworthia plants comprise aglycosyltransferase which is capable of glycosylating flavokermesic acidin order to produce flavokermesic acid glycoside; and/or capable ofglycosylating kermesic acid in order to produce kermesic acid glycoside.

Example 4—GT Activity in Sorghum and Rice Plant

As known the art—Sorghum and rice plants comprise glycosyltransferases.

As known in the art—some of the Sorghum and rice glycosyltransferasesmay glycosylate low molecular weight aglycone compounds.

The in the art described glycosyltransferases from Sorghum and riceplants have significant less than 70% identity with amino acids 1 to 515of SEQ ID NO:2 as disclosed herein.

It is not known in the art if glycosyltransferases of Sorghum and/orrice plants would be a herein relevant glycosyltransferase—i.e. aglycosyltransferase which is capable of glycosylating flavokermesic acidin order to produce flavokermesic acid glycosides; and/or capable ofglycosylating kermesic acid in order to produce kermesic acidglycosides.

The known glycosyltransferases from Sorghum (Sorghum bicolor),SbUGT85B1, with Genbank ID number AF199453.1 (nucleotideseq.)/AAF17077.1 (polypeptide seq) and rice (Oryza sativa), OsCGT, withGenbank ID number FM179712.1 (nucleotide seq.)/CAQ77160.1 (polypeptideseq) were expressed in E. coli strain Xjb and crude E. coli proteinsextracts were prepared and tested for glucosylation activity on thesubstrates kermesic acid and flavokermisic acid as described byKannangara et al. (2011) and Augustin et al. (2012).

FIG. 2 shows LC-MS analyses of glucosylated products formed in assayscontaining crude lysate of E. coli strain Xjb expressing eitherSbUGT85B1 or OsCGT, UDP-glucose and flavokermesicc acid (FK) or kermesicacid (KA). As a negative control crude extract from the E. coli strainXjb was used in the assays.

There were identified KA glycosides (491 m/z [M-H]—the m/z [M-H] valueof CA) for both glycosyltransferases and FK glycosides (475 m/z [M-H]the m/z [M-H] value of DcII) for OsCGT.

Conclusion

The result of this example demonstrated that herein relevantglycosyltransferase (GT) enzymes can be identified in Sorghum and/orrice plants.

Said in other words, Sorghum and/or rice plants comprise aglycosyltransferase which is capable of glycosylating flavokermesic acidin order to produce flavokermesic acid glycoside; and/or capable ofglycosylating kermesic acid in order to produce kermesic acid glycoside.

Example 5—Use of Endogenous GT Gene or GT Activity

As known in the art glycosyltransferases able to glycosylate lowmolecular weight are present in a lot of different organisms. A methodto contact the glycosyltransferase of the cells of an organism with alow molecular weight compound is to introduce one or more genesdirecting the biosynthesis of the low molecular weight compound and thusenabling the cells to glycosylate the low molecular weight compound. Thelow molecular weight compound may be e.g. flavokermesic acid orkermersic acid or decorated versions of these molecules.

One or more genes directing the biosynthesis of flavokermesic acid orkermesic acid or decorated version of these molecules are introducedinto a glycosyltransferase containing organism, e.g. the tobacco plant,Nicotiana benthamiana.

When the gene/genes is/are transiently expressed according to themethods described in D'Aoust et al. (2008) in e.g. plant tissue the lowmolecular weight compound or compounds is/are produced. Cells stablyexpressing the gene/genes are produced and selected according to themethods described in Gelvin (2003).

In cells containing either stably expressed and/or transiently expressedgene/genes the low molecular weight compounds come into contact with theendogenous glycosyltransferases, resulting in the formation of one ormore glycosides of flavokermesic acid, kermesic acid or decoratedversions of these molecules.

The presence of the glycosides is demonstrated by the extraction and theanalytical methods described in example 3.

Samples are prepared for LC/MS by the method for extraction described byRauwald and Sigler (1994).

Conclusion

The results of this example demonstrate that endogenousglycosyltransferases present in the cells of a recombinant organism canbe used to convert flavokermesic acid, kermesic acid or decoratedversions of these molecules into glycosides when a gene/genes directingthe biosynthesis of the aglycons are introduced into the organism.

Said in other words introduction of a gene or genes directing thebiosynthesis of flavokermesic acid, kermesic acid, decorated versions ofthese molecules, or related low molecular weight compounds is a methodto bring the low molecular weight compound in contact withglycosyltransferases and thus a method to produced glycosides offlavokermesic acid, kermesic acid or decorated version of thesecompounds.

REFERENCES

-   1: U.S. Pat. No. 5,424,421 (European Colour, published 1995)-   2: WO2006/056585A1 (Chr. Hansen A/S)-   3: Stathopoulou et al (Analytica Chimica Acta 804 (2013) 264-272)-   4: Zagrobelny et al (Cyanogenic glucosides and plant-insect    interactions; Phytochemistry. 2004 February; 65(3):293-306)-   5: Geuder et al (Journal of Chemical Ecology, Vol. 23, No. 5, 1997)-   6: Genta et al, (Potential role for gut microbiota in cell wall    digestion and glucoside detoxification in Tenebrio molitor larvae),    Journal of Insect Physiology 52 (2006) 593-601-   7: WO2004/111254A1 (Poalis A/S)-   8: Gutmann et al (Pure Appl. Chem, 2013 Jul. 9)-   9: Pompon et al (Methods Enzymol. 272 (1996):51-64)-   10: Baig et al (Angew Chem Int Ed Engl. 2006 Nov. 27; 45(46):7842-6)-   11: Radominska-Pandya A, Bratton S M, Redinbo M R, Miley M J. Drug    Metab Rev. 2010 February; 42(1):133-44)-   12: Plant Physiology, November 2008, Vol. 148, pp. 1295-1308-   13: Esben Halkjaer Hansen et al. Phytochemistry 70(4): 473-482-   14: Kannangara et al. (Plant Journal. 68 (2011): 287-301)-   15: Augustin et al. (Plant Physiology. 160 (2012): 1881-1895)-   16: D'Aoust et al. (Methods Mol Biol 483 (2009): 41-50)-   17: Gelvin (Microbiol Mol Biol Rev 67(1) (2003): 16-37)-   18: Rauwald and Sigler (Phytochemical Analysis 5 (1994):266-270)

The invention claimed is:
 1. A recombinant expression vector comprisinga nucleic acid construct comprising an isolated polynucleotidecomprising a nucleotide sequence which encodes an isolatedglycosyltransferase polypeptide capable of: (I): conjugating nucleotideactivated glucose to flavokermesic acid (FK); and/or (II): conjugatingnucleotide activated glucose to kermesic acid (KA); and wherein theglycosyltransferase polypeptide is at least one polypeptide selectedfrom the group consisting of: (a) a polypeptide comprising an amino acidsequence which has at least 95% identity with amino acids 1 to 515 ofSEQ ID NO:2; and (b) a polypeptide comprising an amino acid sequencewhich has at least 95% identity with amino acids 20 to 468 of SEQ IDNO:2; and wherein said polynucleotide is operably linked to one or morecontrol sequences that direct the production of the polypeptide in anexpression host.
 2. A recombinant host cell comprising the recombinantexpression vector of claim
 1. 3. The recombinant host cell of claim 2,wherein the recombinant host cell is a cell selected from the groupconsisting of a filamentous fungal cell and a microorganism cell, andwherein if the microorganism cell is a yeast cell, the yeast cell isselected from the group consisting of Saccharomyces spp, and Pichia spp.4. The recombinant host cell of claim 3, wherein the recombinant hostcell is Saccharomyces cerevisiae.